Method of producing tooth, set of teeth, and method of producing tissue

ABSTRACT

A first cell mass substantially containing only either one of mesenchymal cells or epithelial cells and a second cell mass substantially containing only the other one of the cells are positioned in contact with each other inside a support carrier which can maintain a condition of cell contact; and cultured to obtain a tooth having a specific cell placement. Preferably, after the culturing, the support carrier having both cell masses is cultured with kidney cells.

FIELD OF THE INVENTION

The present invention relates to a method of producing a tooth, a set ofteeth, and a method of producing tissue and, specifically, to a methodof producing a tooth, a set of teeth, and a method of producing tissueusing cells.

BACKGROUND ART

The tooth is an organ, which can be lost by dental caries, periodontaldiseases or the like, and which has hard tissues such as enamel in theoutermost layer and dentin in the inner layer, and further has anodontoblast which forms dentin in the deeper layer of the tooth anddental pulp in the core. Generally, tooth loss today is mainlycompensated for by dentures and implants in many cases, as this isthought to have little threat to life. However, there is a growinginterest in the development of tooth regenerative technology in view ofthe significant influence that the presence or absence of teeth has onpersonal appearance and on the taste of food, and from the perspectiveof maintaining health and a high quality of life.

Teeth are functional units that are formed by induction during thedevelopmental process of the fetal stage and constructed with pluralcell types, and they are thought to be the same as organs or internalorgans. Therefore, teeth are not produced by the stem cell system inwhich cell types are produced from stem cells such as hematopoietic stemcells and mesenchymal stem cells in the adult body, and teeth cannot beregenerated solely by stem cell implantation (stem cell implant therapy)which is currently under development by regenerative medicine. Moreover,while regeneration of teeth by identifying the gene that is specificallyexpressed in the tooth developmental process and artificially inducing atooth germ is being considered, tooth regeneration cannot be inducedcompletely simply by identifying the gene.

Therefore, studies have been conducted recently with a central focus ontooth regeneration by transplanting a reconstituted tooth germ obtainedby reconstituting a tooth germ using isolated tooth germ cells.

For example, in Non-patent Document 1, it is disclosed that a tooth-liketissue is regenerated by transplanting cells, such as epithelial cellsisolated from a tooth germ and mesenchymal dental follicle cells, with abioabsorbable carrier into an abdominal cavity of a rat.

In Non-patent Document 2, it is described that co-culture by collagengel is effective as a system in which an epithelium/mesenchymalinteraction by subcultured cells can be realized.

As a method of regenerating a tooth germ, it is described, for example,in Patent Document 1, that tooth germ cells are cultured in the presenceof physiologically active substances such as fibroblast growth factorsand the like. In Patent Document 2, it is proposed that at least onetype of cells selected from tooth germ cells and cells which can bedifferentiated into these tooth germ cells are cultured along with afibrin-containing carrier, and it is described that a “tooth” having aspecific shape is formed by using a fibrin-containing carrier having thedesired shape for the tooth germ.

In Patent Documents 3 and 4, a method of forming teeth is disclosed thatincludes seeding a cell mixture of a tooth germ containing dentinforming mesenchymal cells derived from dental pulp and epithelial cellswhich contribute to enamel formation, from the mandible of a 6 month-oldpig, into a scaffold which is a solidified biodegradable polymercontaining a polyglycolic acid/polyacetic acid copolymer; andtransplanting it into an animal body. Here, it is described that a“tooth” having a specific shape is formed by using a scaffoldhaving thedesired shape for the tooth germ.

Further, in Patent Document 5, a method of tooth regeneration fortreating a patient with bone loss or damage is disclosed. According tothis method, a bone is formed by seeding mesenchymal cells in apolyglycolic acid mesh carrier and then laminating the carrier withepithelial cells and collagen or wrapping it with an epithelial cellsheet. Further, in Patent Document 5, a carrier is used to construct theshape of a bone.

Non-patent Document 1: J. Dent. Res., 2002, Vol. 81 (10), pp. 695-700

Non-patent Document 2: “Regenerative medicine using teeth and cellsderived from a tooth germ and the possibility of the same,” RegenerativeMedicine, Journal of the Japanese Society for Regenerative Medicine,2005, Vol. 4(1), pp. 79-83

Patent Document 1: Japanese Patent Application Laid-open No. 2004-331557

Patent Document 2: Japanese Patent Application Laid-open No. 2004-357567

Patent Document 3: US Patent Application Publication No. 2002/0119180

Patent Document 4: US Patent Application Publication No. 2004/0219489

Patent Document 5: International Publication (WO) No. 2005/014070

DISCLOSURE OF THE INVENTION

Problems to be Solved by the Invention

However, in order to function as a tissue, it is essential that pluraltypes of cells constituting a tissue are placed in an appropriaterelative position (cell placement) and have directionality as a tissue.Tissue, for example, a tooth, is an “internal organ” or “organ” producedby an interaction between epithelial cells derived from a tooth germ andmesenchymal cells derived from cranial neural crest cells duringdifferentiation-developmental processes. It is possible to producenormal teeth by transplanting a tooth germ as it is; however, teethhaving the specific cell placement and the directionality of thefunctional unit that is a tooth cannot be regenerated only by isolatingand culturing tooth germ cells constituted by plural types of cells.

Although a tooth germ is reconstructed using cells, cellular factors andthe like in the above-mentioned techniques, the specific cell placementand directionality sufficient to express the functions of a tooth, arenot regenerated.

Further, it has been difficult to reconstruct tissue having the specificcell placement simply by isolating and culturing plural cells whichconstitute the tissue.

Therefore, an object of the present invention is to provide a method ofproducing a tooth having a specific cell placement, a set of toothprepared by this method, and a method of producing periodontal tissue.

Moreover, another object of the present invention is to provide a methodof producing a tissue having a tissue-specific cell placement.

Means for Solving the Problem

A method of producing a tooth of the present invention includespositioning a first cell mass substantially containing only either oneof mesenchymal cells or epithelial cells wherein at least one of themesenchymal cells or epithelial cells is derived from a tooth germ, anda second cell mass substantially containing only the other one of themesenchymal cells or epithelial cells, inside a support carrier and incontact with each other, and culturing the first and the second cellmasses inside the support carrier.

A method of producing periodontal tissue of the present inventionincludes positioning a first cell mass substantially containing onlyeither one of mesenchymal cells or epithelial cells wherein at least oneof the mesenchymal cells or epithelial cells is derived from a toothgerm, and a second cell mass substantially containing only the other oneof the mesenchymal cells or epithelial cells, inside a support carrierand in contact with each other, culturing the first and the second cellmasses inside the support carrier until a tooth and periodontal tissuecontiguous to the tooth are obtained, and isolating the periodontaltissue obtained by the culture.

A set of teeth of the present invention is obtained by positioning afirst cell mass substantially containing only either one of mesenchymalcells or epithelial cells wherein at least one of the mesenchymal cellsor epithelial cells is derived from a tooth germ, and a second cell masssubstantially containing only the other one of the mesenchymal cells orepithelial cells, inside a support carrier and in contact with eachother, and culturing the first and the second cell masses inside thesupport carrier.

In both of the above-mentioned methods or the set of teeth, it ispreferable that each of the above-mentioned first cell mass and secondcell mass is derived from a tooth germ.

Moreover, each of the above-mentioned first cell mass and second cellmass may be a mass of single cells.

Further, a method of producing tissue of the present invention is amethod of producing tissue constructed by interaction betweenmesenchymal cells and epithelial cells, the method including positioninga first cell mass substantially containing only either one ofmesenchymal cells or epithelial cells and a second cell masssubstantially containing only the other one of the mesenchymal cells orepithelial cells, inside a support carrier and in contact with eachother, and culturing the first and the second cell masses inside thesupport carrier.

In the above-mentioned method of producing tissue, it is preferable thatat least one of the above-mentioned mesenchymal cells and epithelialcells is cells derived from a targeted tissue.

Moreover, the above-mentioned tissue is characterized by being selectedfrom the group consisting of a tooth, a hair, a kidney, a lung and aliver.

In the present invention, since cell masses substantially containingonly mesenchymal cells or epithelial cells are positioned inside asupport carrier in contact with each other and cultured, each cell massgrows inside the support carrier without being mixed with the cellswhich constitute the other cell mass, while the state of contact betweenthe masses is maintained. This makes it possible to effectivelyreproduce the excellent interaction between the mesenchymal cells andthe epithelial cells required in formation of the tissue.

As a result, a tissue having the specific cell placement for thetargeted tissue can be prepared. Moreover, a tooth or a set of teethhaving a specific cell placement, in which there is enamel outside anddentin inside, can be prepared when at least one of the mesenchymalcells and the epithelial cells is derived from a tooth germ.

Effect(s) of the Invention

According to the present invention, a method of producing a tooth havinga specific cell placement, a set of teeth prepared by this method, and amethod of producing periodontal tissue can be provided.

Furthermore, according to the present invention, a method of producingof tissue having cell placement specific to the tissue can be provided.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 is a schematic diagram showing formation of the tooth germ.

FIG. 2 (A) to (D) are schematic views conceptually showing a procedurefor reconstruction of a tooth germ using mesenchymal cells andepithelial cells which are derived from a tooth germ, according to theExamples of the present invention.

FIG. 3 shows phase contrast images and staining images of normal toothgerm tissues and time course staining images of the tooth produced bythe subrenal capsule transplantation of the normal tooth germ, accordingto Comparative Example 1 of the present invention.

FIG. 4 shows phase contrast images of a tooth germ reconstituted byepithelial tissues derived from a tooth germ and mesenchymal cellsderived from a tooth germ and time course staining images of a toothproduced by subrenal capsule transplantation of the reconstituted toothgerm, according to Example 1 of the present invention.

FIG. 5 shows phase contrast images of a tooth germ reconstituted byepithelial tissue derived from a tooth germ and mesenchymal cellsderived from a tooth germ of a GFP mouse and a staining image of the14^(th) day of the tooth produced by subrenal capsule transplantation ofthe reconstituted tooth germ, according to Example 1 of the presentinvention.

FIG. 6 shows phase contrast images of a tooth germ reconstituted byepithelial cells derived from a tooth germ of a GFP mouse andmesenchymal tissues derived from a tooth germ and a staining image ofthe 14^(th) day of the tooth produced by subrenal capsuletransplantation of the reconstituted tooth germ, according to Example 2of the present invention.

FIG. 7 shows phase contrast images of a tooth germ reconstituted byepithelial cells derived from a tooth germ and mesenchymal cells derivedfrom a tooth germ and a staining image of the 14^(th) day of the toothproduced by subrenal capsule transplantation of the reconstituted toothgerm, according to Example 3 of the present invention.

FIG. 8 shows phase contrast images of epithelial tissues derived from atooth germ and mesenchymal tissues derived from a tooth germ andchromatic figures of the 14^(th) day of individual subrenal capsuletransplantation of each of the above tissues, according to ComparativeExample 2 of the present invention.

FIG. 9 shows phase contrast images of a low density tooth germreconstituted by using epithelial tissues derived from a tooth germ andmesenchymal cells derived from a tooth germ and staining images of the20^(th) day of subrenal capsule transplantation of the low densityreconstituted tooth germ, according to Comparative Example 3 of thepresent invention.

FIG. 10 shows phase contrast images of a tooth germ reconstituted byreconstituting epithelial tissues derived from a tooth germ andmesenchymal cells derived from a tooth germ in high density and withoutcompartmentalization, and staining images of the 20^(th) day of subrenalcapsule transplantation of the low density reconstituted tooth germaccording to Comparative Example 4 of the present invention.

FIG. 11 shows staining images of the alveolar bone and periodontalmembrane which are periodontal tissues formed around the tooth producedfrom the reconstituted tooth germ according to Examples 1 to 3 of thepresent invention.

FIG. 12 shows staining images of detected periostin mRNA specific to aperiodontal membrane which is a periodontal tissue formed around thetooth produced from the reconstituted tooth germ according to Example 2of the present invention (17^(th) day after the transplantation) andComparative Example 1 (14^(th) day after the transplantation).

FIG. 13 shows phase contrast images and staining images of the toothproduced by organ culture by extending the culture process afterpreparing the reconstituted tooth germ according to Examples 4 and 5 ofthe present invention and Comparative Example 5.

FIG. 14 shows phase contrast images of a tooth germ reconstituted byepithelial cells derived from a tooth germ and mesenchymal cells derivedfrom a tooth germ and staining images of the 14^(th) day of the toothproduced by subrenal capsule transplantation of the reconstituted toothgerm, according to Example 6 of the present invention.

FIG. 15 (A) is a staining image of a non-transplant mouse on the 14^(th)day after tooth extraction of the present invention and (B) is anenlarged view of the area enclosed by the frame of (A), according toComparative Example 6.

FIG. 16 shows staining images of the 14^(th) day after transplantationof an individually separated tooth germ into the oral cavity, accordingto Example 6 of the present invention.

FIG. 17 shows staining images of the 14^(th) day after transplantationof an individually separated tooth into the oral cavity, according toExample 7 of the present invention.

FIG. 18 shows phase contrast images of a hair follicle reconstituted byepithelial cells derived from hair follicle tissue and mesenchymal cellsderived from hair follicle tissue and staining images of the 14^(th) dayof the hair produced by subrenal capsule transplantation of thereconstituted hair follicle, according to Example 8 of the presentinvention.

FIG. 19 shows stereoscopic microscope images of the 14^(th) day of thehair follicle produced by subrenal capsule transplantation of thereconstituted hair follicle from epithelial cells derived from hairfollicle tissue and mesenchymal cells derived from hair follicle tissueaccording to Example 8 of the present invention.

FIG. 20 shows phase contrast images of a hair follicle reconstituted byepithelial cells derived from hair follicle tissue and mesenchymal cellsderived from hair follicle tissue according to Comparative Example 7 ofthe present invention and staining images of the 14^(th) day of the hairfollicle produced by subrenal capsule transplantation of thereconstituted hair follicle.

BEST MODE FOR CARRYING OUT THE INVENTION

A method of producing a tooth of the present invention includes:positioning a first cell mass substantially containing only either oneof mesenchymal cells or epithelial cells in which at least one of themesenchymal cells or epithelial cells is derived from a tooth germ and asecond cell mass substantially containing only the other one of themesenchymal cells or epithelial cells, inside a support carrier and incontact with each other (placement process); and culturing theabove-mentioned first and second cell masses inside the above-mentionedsupport carrier (culture process).

In the present method of producing a tooth, since the mesenchymal cellsand epithelial cells, at least one of the mesenchymal cells orepithelial cells being derived from a tooth germ, are grown as cellmasses in a support carrier while in contact with each other, theinteraction between the cells can be effectively performed due to thestate of close contact therebetween and a tooth having a cell placementspecific to teeth, in which there is dentin inside and enamel outside,can be produced.

In the present invention, the term, “tooth” refers to a tissue having adentin layer inside and an enamel layer outside contiguously, andpreferably to a tissue having these layered structures and also adirectionality having a crown and root. Those skilled in the art caneasily identify dentin and enamel morphologically by tissue staining andthe like. Moreover, enamel can be identified by the presence of anameloblast, and the presence of an ameloblast can be confirmed by thepresence of amelogenin. On the other hand, dentin can be identified bythe presence of an odontoblast, and the presence of an odontoblast canbe confirmed by the presence of dentin sialoprotein. The presence ofamelogenin and dentin sialoprotein can be confirmed easily by awell-known method in the art; for example, in situ hybridization,antibody staining or the like.

Further, the directionality of a tooth can be identified by theplacement of a crown and root. The crown and root can be confirmed basedon the visually observed shape and tissue staining.

In addition, in the present invention, the term “periodontal tissue”refers to an alveolar bone and a periodontal membrane formed mainly inthe outer layer of a tooth. Alveolar bone and periodontal membrane canbe easily morphologically identified by those skilled in the art bytissue staining or the like.

Further, in the present invention, the term, “mesenchymal cells” refersto cells derived from mesenchymal tissue and “epithelial cells” refersto cells derived from epithelial tissue.

In the present invention, the terms, “tooth germ” and “tooth bud” areexpressions used to refer specifically to the tooth germ and tooth budwhich are distinguishable from other tissue based on the developmentalstage described later. In this case, “tooth germ” refers to anearly-stage embryo of a tooth, which is destined to become a tooth inthe future, and to tissue from the bud stage to the bell stage in thetypical developmental stages of a tooth and, specifically, to tissue atwhich no accumulation of dentin and enamel is identified, which are thefeatures of tooth as a hard tissue. A “tooth bud” refers to a tissue interms of the transition of the stages of the “tooth germ” used in thepresent invention, and to a tissue between the stage where theaccumulation of dentin and enamel, which are the features of the hardtissue of tooth, is started, and the stage before the tooth germinatesfrom the gum to manifest the typical functions of the tooth.

A tooth germ, as shown in FIG. 1, develops through each stage of a budstage, a cap stage, an early bell stage and a late bell stage in theontogenic process. In the bud stage, epithelial cells invaginate to wraparound mesenchymal cells (see (A) and (B) of FIG. 1), and the epithelialcell portion becomes the outer enamel and the mesenchymal cell portionbegins to form dentin internally (see (C) and (D) of FIG. 1) as it movesto the early bell stage and late bell stage. Therefore, a tooth germ isformed by the interaction between epithelial cells and mesenchymalcells.

The mesenchymal cells and epithelial cells in the present invention maybe those in the stages from the above-mentioned bud stage to the latebell stage, where a tooth germ is formed or can be formed (hereinafter,simply referred to as a “tooth germ”), and from the viewpoint of thelevel of immaturity and the homogeneity in the differentiation stages ofthe cells, it is preferable that they are in the stages from the budstage to the cap stage.

Moreover, the term, “cell mass” refers to a state in which cells areclosely packed and may refer to the condition of tissue or the conditionof single cells. In addition, the term, “substantially containing” meansthat anything other than the target cells are excluded to the greatestpossible extent. Since each cell mass may be a tissue itself or a partthereof, or a mass of single cells, either one of the cell masses may bea cell mass constituted by single cells or both of the cell masses maybe cell masses constituted by single cells; however, in order toeffectively achieve the reconstruction of tissue according to thepresent invention, it is preferable that both cell masses areconstituted by single cells.

Either a first cell mass or a second cell mass may be epithelial cellsor mesenchymal cells and the number of cells constituting the cell massmay vary depending on animal species, and on the type, hardness and sizeof the support carrier, but it may generally be from 10¹ to 10⁸ cellsper cell mass, and preferably from 10³ to 10⁸ cells per cell mass.

In the positioning process, a first cell mass and a second cell mass arepositioned inside a support carrier in contact with each other.

In the positioning process of the production method of the presentinvention, since the above-mentioned first and second cell masses arepositioned inside a support carrier which can maintain the state ofcontact of the cells, cells constituting each cell mass do not mix withthe cells constituting the other cell mass. Thus, in the positioningprocess, each cell mass is positioned without being mixed with theother, and a boundary surface is formed between the cell masses. Such anpositioning mode is suitably expressed as “compartmentalization” in thepresent specification.

In this case, a first cell mass and a second cell mass are prepared inindependent preparation processes (first cell preparation process andsecond cell preparation process) so that each of cell mass may besubstantially constituted by mesenchymal cells or epithelial cells.

At least either one of mesenchymal cells or epithelial cells used in thepresent production method may be derived from a tooth germ in order toreproduce in vivo cell placement to form a tooth having a specificstructure and directionality; however, in order to ensure toothproduction, most preferably both of the mesenchymal cells and theepithelial cells are derived from a tooth germ.

Examples of mesenchymal cells derived from other than a tooth germinclude cells derived from other mesenchymal tissues in vivo;preferably, bone marrow cells including no blood cells, or mesenchymalstem cells; more preferably, mesenchymal cells in the oral cavity andbone marrow cells inside the jaw bone, and mesenchymal cells derivedfrom cranial neural crest cells; and mesenchymal precursor cells, whichcan generate the above-mentioned mesenchymal cells, stem cells thereof,and the like.

Further, examples of epithelial cells derived from tissues other than atooth germ include cells derived from other epithelial tissues in vivo;preferably, epithelial cells of skin, mucous membrane or gum in the oralcavity; and more preferably, immature epithelial precursor cells whichcan produce differentiated epithelial cells, for example, keratinized orparakeratinized epithelial cells of the skin and mucous membrane; forexample, non-keratinized epithelial cells and stem cells thereof, andthe like.

The tooth germ and other tissues may be collected from the jaw bone ofvarious animals such as primate mammals such as humans and monkeys;ungulates such as pigs, cows and horses; rodent small mammals such asmice, rats, and rabbits. In the collection of the tooth germ and tissue,conditions generally used in the collection of tissue may be appliedwithout modification, and the tooth germ and tissue may be extracted insterile conditions and stored in an appropriate preservative solution.Further, examples of a human tooth germ include a fetal tooth germ aswell as the third molar, or so-called wisdom tooth, but it is preferableto use the tooth germ of a wisdom tooth from the standpoint of the useof autogenous tissues.

The preparation of mesenchymal cells and epithelial cells from such atooth germ is started by separating a tooth germ, which has beenisolated from the surrounding tissue, into a tooth germ mesenchymaltissue and a tooth germ epithelial tissue according to the respectiveshapes thereof. The tooth germ tissues can be easily separated bycutting or tearing using dissecting scissors, tweezers or the like,since it is possible to identify the tooth germ tissues structurallyunder a microscope. Further, the separation of tooth germ mesenchymaltissue and tooth germ epithelial tissue from the tooth germ can beeasily done by cutting or tearing using injection needles, tungstenneedles, tweezers or the like, according to the respective shapesthereof.

Preferably, enzymes may be used to easily separate tooth germ cells fromthe surrounding tissue and/or to separate an epithelial tissue and amesenchymal tissue from a tooth germ tissue. Examples of the enzymesused in such applications include dispase, collagenase, trypsin and thelike.

Mesenchymal cells and epithelial cells may be prepared in a single cellstate from a mesenchymal tissue and an epithelial tissue, respectively.In the preparation process, enzymes may be used to make the cells easilydispersible as single cells. Examples of such enzymes include dispase,collagenase, trypsin and the like. In this case, for separation of theepithelial cells from the epithelial tissue, it is preferable to performtrypsin treatment and DNase treatment after collagenase treatment. Onthe other hand, for the separation of the mesenchymal cells from themesenchymal tissue, it is preferable to perform collagenase treatmentand trypsin treatment simultaneously and ultimately to perform DNasetreatment. In this case, the DNase treatment is performed in order toprevent a decrease in the amount of recovered cells resulting from cellaggregation caused by DNA released into the solution when the cellmembrane is lysed, after some of the cells are damaged by the enzymetreatments.

In addition, the mesenchymal cells and epithelial cells may be thosewhich have been subjected to preliminary culture prior to thepositioning process in order to obtain a sufficiently large number ofeach kind of the cells. In the culture of mesenchymal cells andepithelial cells, the usual conditions, such as temperature, used in theculture of animal cells can be applied without modification.

As a medium used in the culture, a medium generally used for animal cellculture, such as Dulbecco's Modified Eagle Medium (DMEM), can be used.Serum may be added to promote cell proliferation, or as alternatives tothe serum, cellular growth factors such as FGF, EGF, PDGF and the likeor well-known serum components such as transferin may be added. Inaddition, when serum is added, the concentration of the serum may bechanged appropriately depending on the culture conditions, but it canusually be 10%. For cell culture, the culture conditions generally usedin culture, such as of culture in an incubator in 5% CO₂ concentrationat 37° C., may be applied. Moreover, antibiotics such as streptomycinmay be added as appropriate.

As a support carrier used in the present invention, a support carrier inwhich cells can be cultured may be used, and a mixture with theabove-mentioned culture medium is preferable. Examples of such supportcarriers include collagen, fibrin, laminin, an extra-cellular matrixmixture, polyglycolic acid (PGA), polylactic acid (PLA), lacticacid/glycolic acid copolymer (PLGA), Cellmatrix (trade name), Mebiol Gel(trade name), and Matrigel (trade name). These support carriers may beof a hardness that can virtually maintain the approximate location atwhich the cells are positioned inside thereof, and examples thereofinclude gel type, fiber type, and solid type carriers. In this case, thelevel of hardness that can maintain the location of the cells may be thelevel of hardness generally applied in three-dimensional culture; inother words, a level of hardness that does not inhibit hypertrophy ofthe cells due to proliferation while maintaining the positioning of thecells, and the level of hardness is easily determined. For example, inthe case of collagen, use at a final concentration of 2.4 mg/ml providesan appropriate level of hardness.

In addition, in this case, the support carrier may be of a thicknesssufficient for the first and second cell masses to grow inside thecarrier and is set appropriately based on the size of the targetedtissues.

Moreover, the support carrier may be one that can maintain the state ofcontact between the cells. The “state of contact” as referred to hereinis preferably a high density state in order to ensure cell interactionwithin each cell mass or between the cell masses.

A high density state refers to a density similar to that at the timewhen the tissue is constituted such as, in the case of the cell masses,5×10⁷ to 1×10⁹/ml at the time of cell placement, preferably 1×10⁸ to1×10⁹/ml to ensure cell interaction without sacrificing the cellactivity, and most preferably 2×10⁸ to 8×10⁸/ml. In order to prepare acell mass at such a cell density, it is preferable to aggregate cellscentrifugally and have these precipitated, since this convenientlyenables high density without sacrificing the cell activity. Thecentrifugation may be performed at a revolution speed equivalent to acentrifugal force of 300 to 1200×g, which will not preclude cellsurvival, and preferably 500 to 1000×g, for 3 to 10 minutes.Centrifugation at lower than 300×g may lead to insufficient cellprecipitation and the cell density may become low, while centrifugationat higher than 1200×g may lead to cell damage and, therefore, neither ofthese cases is preferable.

When high density cells are prepared by centrifugation, centrifugationis generally performed after preparing a suspension of single cells incontainers such as tubes used for cell centrifugation, and thesupernatant is removed to the greatest extent possible, leaving cells asthe precipitate. It is preferable that the containers such as tubes aresilicon-coated from the standpoint of completely removing thesupernatant.

When precipitates are prepared by centrifugation, the precipitates maybe directly positioned inside the support carrier. Here, componentsother than the targeted cells (for example, a culture solution, a buffersolution, the support carrier and the like) are preferably equal to orless in volume than the cells, and most preferably the components otherthan the targeted cells are excluded. In such a high density cell mass,cells are in close contact with each other and the interaction betweenthe cells may be achieved effectively.

When used in a tissue state, it is preferable to remove components otherthan the target cells, such as connective tissues, by performing anenzyme treatment or the like. When there are many components other thanthe target cells, for example, when the volume of the other componentsis equal to or more than that of the cells, the interaction between thecells may not be achieved sufficiently, and this is not preferable.

Moreover, it is more preferable when a first cell mass and a second cellmass are in very close contact, and it is especially preferable toposition the second cell mass so as to press against the first cellmass. Furthermore, encompassing the surroundings of the first cell massand the second cell mass with a culture solution or solid which does notinhibit oxygen permeation is also effective in making the contactbetween the cell masses closer, and it is also preferable to add andposition a cell suspension at high density into a solution with adifferent viscosity to solidify the solution as is, since the cellcontact can be easily maintained thereby. Here, it is preferable toposition the enamel knot of a tooth germ epithelial tissue in contactwith the first cell mass when the first cell mass is single cell mass oftooth germ mesenchymal cells and the second cell mass is a tooth germepithelial tissue, but the invention is not limited to this.

When the support carrier is in a gel state, a solution state or thelike, the solidification process by which a support carrier issolidified may be arranged so as to follow after the positioningprocess. Cells positioned inside the support carrier may be fixed insidethe support carrier by the solidification process. For solidification ofthe support carrier, conditions generally used for the solidification ofsupport carriers may be applied without modification. For example, whensolidifiable compounds such as collagen are used for the supportcarrier, they can be solidified under generally applied conditions by,for example, being held still for several minutes to several tens ofminutes at the culture temperature. In this way, binding between cellsinside the support carrier can be fixed and robust.

In the culture process of the production method of the presentinvention, a first cell mass and a second cell mass are cultured insidethe support carrier. In this culture process, the interaction betweenthe cells is effectively performed by the first cell mass and the secondcell mass which are in close contact with each other, to reconstitute atissue, namely, a tooth.

The culture process may be performed such that the state of contactbetween the first cell mass and the second cell mass is maintained bythe support carrier and the process may be culture in a support carrierwhich simply has first and second cell masses, or culture in thepresence of other cells of animals.

The culture period varies depending on the number of cells positioned inthe support carrier and the state of the cell mass and, further, on theconditions under which the culture process is performed; however, ittypically takes from 1 to 300 days, and preferably from 1 to 120 days,to form a tooth having enamel outside and dentin inside, and preferably1 to 60 days from the standpoint of providing quick results. Further, ittypically takes 1 to 300 days, and preferably 1 to 60 days, to form atooth having periodontal tissue.

When culture is performed only in the support carrier, culture can beperformed under the general conditions used for culture of animal cells.Here, conditions for culture generally used for animal cells can beapplied without modification and the above-mentioned conditions can beapplied without modification. Further, serum derived from mammals andvarious cellular factors which are known to be effective inproliferation and differentiation of these cells, may be added to theculture. Examples of these cellular factors include FGF and BMR.

In addition, it is preferable to use organ culture from the standpointof gas exchange and nutrient supply for tissues and cell masses. Inorgan culture, generally, culture is performed by floating a porousmembrane on a culture medium suitable for proliferation of animal cellsand placing the cell masses embedded in a support carrier on themembrane. The porous membrane used herein is preferably a membranehaving many pores of 0.3 to 5 μm in diameter and specific examplesinclude a Cell Culture Insert (trade name) and an Isopore Filter (tradename).

Performing the culture in the presence of other cells of animals ispreferable because a tooth having a specific cell placement can beformed in the early stage in response to the actions of variouscytokines and the like from animal cells. Such culture in the presenceof other cells of animals may be performed ex vivo using isolated cellsand cultured cells.

Furthermore, it is especially preferable to perform culture in vivo bytransplanting the support carrier having first and second cell massesinto a living body, since a tooth and/or a periodontal tissue can beformed in an early stage. In this case, the first and the second cellmasses are transplanted with the support carrier into the living body.

Animals which can be used for this application preferably includemammals, for example, humans, pigs, mice and the like, and morepreferably animals derived from the same species as that of the toothgerm tissue. When a human tooth germ tissue is transplanted, it ispreferable to use a human or mammals other than humans which have beenaltered to be immunodeficient. As for sites in a living body suitablefor such in vivo growth, subrenal capsule, mesentery, and subcutaneoustransplantation are preferable for the transplantation in order togenerate organs or tissues of the animal cells as normally as possible.

The growth period according to the transplantation varies depending onthe size of the explant at the time of transplantation and the size ofthe tooth to be produced, but is typically 3 to 400 days. For example,the subrenal capsule transplantation period is preferably 7 to 60 daysfrom the standpoint of the tooth regeneration and the size of the toothto be produced at the site of the transplantation, although it variesdepending on the size of explant to be transplanted and the size of thetooth to be regenerated.

Ex vivo culture (preculture) may be performed prior to transplantationto the living body. The preculture is preferable since the bonds betweencells and the bond between the first and the second cell masses can bemade stronger to make the interaction between cells stronger. As aresult, the overall growth period can be shortened.

The preculture period may be short or long. It is desirable to have alonger period, for example, 3 days or more, and preferably 7 days ormore, since a tooth bud can be produced from a tooth germ and thus theperiod until a tooth is formed after the transplantation can beshortened. The period of preculture of, for example, organ culture totransplant beneath a subrenal capsule, is preferably 1 to 7 days inorder to effectively regenerate a tooth.

A tooth produced according to the production method of the presentinvention has a tooth-specific cell placement (structure) having dentininside and enamel outside, and preferably has directionality, that is,has a tip (crown) and a root of a tooth. By having at least thisspecific cell placement, and by preferably having directionality inaddition to the cell placement, the functions of a tooth can bemanifested. Therefore, such a tooth can be widely used extensively as atooth replacement. Particularly when the mesenchymal cells andepithelial cells derived from an autogenous tooth germ are used,problems caused by rejection can be avoided. Generally, it is alsopossible to avoid problems caused by rejection when the cells arederived from the tooth germ of another human having a matchingtransplantation antigen.

Further, in the present invention it is possible to form periodontaltissue in addition to a tooth itself, such as alveolar bone andperiodontal membrane, which support and stabilize teeth on the jaw bone,by extending the culture period. As a result, a practicable tooth can beprovided after the transplantation.

That is, the method of producing periodontal tissue of the presentinvention is characterized by containing the above-mentioned cultureprocess as a step to culturing the above-mentioned first and second cellmasses inside a support carrier until a tooth and a periodontal tissuecontiguous to the tooth can be obtained (culture process), and furthercontaining a step to isolate the periodontal tissue obtained by theabove-mentioned culture.

In this method, a periodontal tissue can be formed contiguously to thetooth by extending the culture period until the periodontal tissue isobtained, and periodontal tissue can be obtained in isolation byseparating it from the tooth. Isolation of the periodontal tissue may beperformed according to any method, in which the periodontal tissueformed during the culture process can be separated from a tooth, forexample, separation by tweezers or the like, partial digestion byenzymes, or the like.

In addition, anything described in the above-mentioned method ofproducing a tooth can be applied to the present method of producing aperiodontal tissue as long as the culture period is not limited.

The tooth and periodontal tissue obtained by the above-mentioned methodof producing a tooth and method of producing periodontal tissue of thepresent invention can be used as an effective research tool forproducing tissue related to teeth in the future since, in addition touse as an explant, they can be applied to studies investigating thedevelopmental processes of teeth.

Moreover, when the tooth or periodontal tissue obtained is used as anexplant, the culture process according to the production method ispreferably performed in organ culture in which there is no contact withother cells of animals and the entire procedure can be processed invitro.

A set of teeth of the present invention is a set of teeth having atooth-specific cell placement obtained by the above-mentioned method ofproducing a tooth.

Since such a set of teeth is constituted by plural teeth having atooth-specific cell placement, each individual tooth can be separatedfrom the set of teeth and used as an explant of a single tooth asdescribed below. Thus, in the method of producing a tooth of the presentinvention, when plural teeth are produced simultaneously, the teeth canbe provided as a set of teeth constituted by plural teeth. As a result,teeth for explants can be efficiently produced.

The set of teeth can be easily obtained by applying the above-mentionedmethod of producing a tooth without modification. In particular, in theabove-mentioned first and second cell preparation processes, each of afirst and a second cell mass is separately prepared and then positionedinside a support carrier in contact with each other in the positioningprocess and, therefore, plural teeth can easily be formed from a cellgroup which normally forms only a single tooth.

In the method of producing a set of teeth, it is preferable that both ofa first and a second cell mass are constituted by single cells in orderto facilitate the reinduction of tooth germs to produce plural teeth. Inaddition, the culture process may be either organ culture or subrenalcapsule culture as mentioned above, and when the obtained tooth is usedas an explant, it is preferable to perform organ culture in which thereis no contact with other cells of animals and the entire procedure canbe processed in vitro.

Furthermore, a tooth transplantation method is included in the presentinvention. This transplantation method includes: a process to obtain theabove-mentioned set of teeth; a process in which each tooth is separatedfrom a set of teeth; and a process in which a separated tooth is alignedto have the same directionality as other teeth at the transplantationsite, and transplanted.

Thereby, plural teeth having a specific cell placement anddirectionality can be obtained simultaneously and tooth transplantationcan be performed efficiently.

The tooth according to the present invention can be applied totreatments or procedures for tooth loss caused by various symptomsaccompanied by loss of or damage to teeth: for example, dental caries,marginal periodontitis (alveolar pyorrhea), and periodontal diseases,tooth breakage or avulsion caused by accidents, and the like.

In other words, a treatment method of the present invention includestransplantation of the tooth and/or periodontal tissue obtained by theproduction method of the present invention into the site of tooth lossand/or damage. Thereby, the above-mentioned symptoms at the site oftooth loss and/or damage can be treated and/or alleviated.

Another treatment method of the present invention includes carrying outonly the culture process of the present invention, or carrying out thepositioning process and culture process at the site of tooth loss and/ordamage. In this case, the surrounding tissue at the site of tooth lossand/or damage itself may be applied as a support carrier in addition tothe support carriers mentioned above. Thus, the site of tooth lossand/or damage can be treated faster by cytokine and the like from thesurrounding tissues in the living body.

In the present invention, since tissue can be effectively reconstitutedby an interaction between mesenchymal cells and epithelial cells, amethod of producing tissue which is constructed by an interactionbetween mesenchymal cells and epithelial cells can also be provided.

In other words, the method of producing a tissue of the presentinvention is a method of producing tissue constructed by an interactionbetween mesenchymal cells and epithelial cells, and includes:positioning a first cell mass substantially containing only either oneof mesenchymal cells or epithelial cells and a second cell masssubstantially containing only the other of the mesenchymal cells orepithelial cells inside a support carrier in contact with each other;and culturing the above-mentioned first and second cell masses insidethe above-mentioned support carrier.

In addition, items described in the above-mentioned method of producinga tooth may be similarly applied to the present method of producingtissue, unless otherwise noted.

As tissues produced by the present method of producing tissue, thoseconstructed by the interaction between mesenchymal cells and epithelialcells are pertinent, and examples of these include a hair, kidney, lung,liver or the like in addition to the tooth mentioned above, and mayinclude the entire tissue or a part thereof.

In this case, it is preferable that at least one of the mesenchymalcells and epithelial cells is derived from the target tissue. In thisway, a tissue can be easily formed by using cells which have alreadybeen directed to the target tissue. Moreover, in order to produce atargeted tissue more reliably, it is most preferable that both of themesenchymal cells and epithelial cells are derived from the targettissue.

Examples of tissues used to prepare a cell mass respectively constitutedby mesenchymal cells or epithelial cells include: in the case of atooth, a tooth germ and dental pulp cells, periodontal membrane cells,and epithelial/mesenchymal cells in the oral cavity; in the case ofhair, a primordial hair follicle in the developmental process and a hairfollicle tissue of an adult; in the case of a kidney, a primordialkidney in the developmental process and a kidney tissue of an adult; inthe case of a lung, a primordial lung in the developmental process and alung tissue of an adult; and in the case of a liver, a primordial liverin the developmental process and a liver tissue of an adult.

In order to prepare each cell mass from these tissues, a first and asecond cell mass may be prepared as described above by separatingmesenchymal cells and epithelial cells from a tissue, positioning theminside a support carrier, and culturing and/or transplanting asdescribed above.

In this way, as with the above-mentioned tooth, a tissue having aspecific cell placement for the targeted tissue can be obtained.

The followings are explanations of Examples of the present invention,but the present invention is not limited to these. “%” as used inExamples is based on weight (mass), unless otherwise noted.

EXAMPLES Examples 1 to 3 and Comparative Examples 1 to 4

(1) Preparation of Tooth Germ Epithelial Cells and Tooth GermMesenchymal Cells

A tooth germ was reconstructed to form a tooth. Mice were used as themodel for this experiment.

A mandibular incisor tooth germ tissue was excised from an embryo,having an embryonic age of 14.5 days, of a C57BL/6N mouse (purchasedfrom CLEA Japan, Inc.) or a C57BL/6-TgN (act-EGFP) OsbC14-Y01-FM131 (GFPmouse: RIKEN Bioresource Center) which is a Green Fluorescence Protein(EGFP) transgenic mouse, by the conventional method under a microscope.The mandibular incisor tooth germ tissue was washed with a phosphatebuffer solution (PBS (−)) containing neither Ca2+ nor Mg2+, treated withan enzyme solution, in which Dispase II (Roche, Mannheim, Germany) wasadded to the PBS (−) at a final concentration of 1.2 U/ml, at roomtemperature for 12.5 minutes, and then washed three times with DMEM(Sigma, St. Louis, Mo.) to which 10% of FCS (JRH Biosciences, Lenexa,Kans.) had been added. Furthermore, a DNase I solution (Takara, Shiga,Japan) was added to make the final concentration 70 U/ml and the toothgerm tissue dispersed, and tooth germ epithelial tissues and tooth germmesenchymal tissues were surgically separated using a 25 G injectionneedle (Terumo, Tokyo, Japan).

For tooth germ epithelial cells, the tooth germ epithelial tissueobtained above was washed three times with PBS (−), and treated twicewith an enzyme solution, in which Collagenase I (Worthington, Lakewood,N.J.) at a final concentration of 100 U/ml was dissolved in the PBS (−),at 37° C. for 20 minutes. The cells precipitated and retrieved bycentrifugation were further treated with 0.25% Trypsin (Sigma)-PBS (−)at 37° C. for 5 minutes. After washing the cells three times with DMEMsupplemented by 10% FCS, a DNase I solution at a final concentration of70 U/ml was added to the cells, and single tooth germ epithelial cellswere obtained by pipetting.

On the other hand, for tooth germ mesenchymal cells, the tooth germmesenchymal tissue was washed three times with PBS (−) and treated withPBS (−) containing 0.25% Trypsin (Sigma) and 50 U/ml of Collagenase I(Worthington). 70 U/ml of DNase I (Takara) was added and single toothgerm mesenchymal cells were obtained by pipetting.

(2) Preparation of Reconstituted Tooth Germ

Next, a tooth germ was reconstructed using the tooth germ epithelialcells and tooth germ mesenchymal cells prepared as above.

Tooth germ epithelial cells or tooth germ mesenchymal cells suspendedwith DMEM (Sigma) supplemented by 10% FCS (JRH Biosciences), were addedto a silicon grease coated 1.5 mL microtube (Eppendorf, Hamburg,Germany), and the cells were retrieved as precipitates by centrifugation(580×g). The supernatant of the culture solution after centrifugationwas removed to the greatest extent possible, centrifugation wasconducted again, and the culture solution remaining around cellprecipitates was completely removed using a GELoader Tip 0.5 to 20 μL(Eppendorf) while being observed under a stereomicroscope to preparecells to use for generating a reconstituted tooth germ.

30 μL of Cellmatrix type I-A (Nitta Gelatin, Osaka, Japan) prepared withthe above-mentioned culture solution at a concentration of 2.4 mg/ml wasdropped on a silicon grease coated Petri dish to generate a drop (geldrop) of collagen solution. 0.2 to 0.3 μL of the precipitates fromcentrifugation of the above-mentioned tooth germ epithelial cells ortooth germ mesenchymal cells were applied to this solution using apipette tip (Quality Scientific Plastics) of 0.1 to 10 μL to generatecell aggregates as cell masses.

This will be explained with reference to FIG. 2.

Cell aggregate 12, which was first placed inside gel drop 10 usingpipette tip 16, configures a sphere inside the gel drop 10 (see FIG. 2(B)). When another cell aggregate 14 is then inserted, the sphericalcell aggregate 12 is crushed and envelops the cell aggregate 14 in manycases (see FIG. 2 (C)). Then, by solidifying the gel drop 10, the bondsbetween cells are strengthened.

In the present Example, a cell aggregate containing single cells of theepithelial cells or mesenchymal cells, and a partial tissue containingepithelial cells and a partial tissue containing mesenchymal cells of atooth germ were prepared, respectively, as cell masses and used.

In the present Example, when combining the reconstituted tooth germsobtained from a cell aggregate and a tissue (Examples 1 and 2), aftertransferring the partial tissue containing epithelial cells ormesenchymal cells into a gel drop, a boundary surface of the tooth germof each tissue was placed in close contact with a cell aggregategenerated from tooth germ epithelial cells or mesenchymal cells using atungsten needle to generate a reconstituted tooth germ.

Further, for the reconstituted tooth germ (Example 3) using tooth germepithelial cells and tooth germ mesenchymal cells which were made assingle cells, a cell aggregate was prepared by applying the tooth germepithelial cells in a similar way to the above so as to contact with thecell aggregate of the tooth germ mesenchymal cells prepared in advance,and a reconstituted tooth germ was prepared such that both would be inclose contact with each other.

A reconstituted tooth germ prepared inside a gel drop was set still in aCO₂ incubator for 10 minutes to solidify the Cellmatrix type I-A (NittaGelatin), and a cell aggregate along with the surrounding gel as asupport carrier, were transferred onto a membrane of a cell cultureinsert in a culture vessel which was arranged such that the cell cultureinsert (PET membrane with a pore size of 0.4 microns; BD, FranklinLakes, N.J.) was in contact with DMEM (Sigma) supplemented by 10% FCS(JRH), and organ cultured for 18 to 24 hours. After the organ culture,the tooth generation was analyzed by promoting ectopic tooth generationafter transplanting the explant along with the surrounding gel beneath asubrenal capsule of an 8 week-old C57BL/6 mouse, or by continuing theorgan culture on the cell culture insert.

As comparative examples, each of the following was prepared and analyzedin the same manner as above: an explant transplanted with an entiretooth germ tissue beneath a subrenal capsule (Comparative Example 1); anexplant respectively transplanted with each of the epithelial tissue andmesenchymal tissue individually separated from a tooth germ (ComparativeExample 2); an explant using a low-density aggregate containing anamount of a culture solution equal to the volume of the cells(Comparative Example 3); and an explant formed from a cell aggregateinside a support carrier by mixing epithelial cells and mesenchymalcells separated from a tooth germ without compartmentalization betweenthe epithelial cells and the mesenchymal cells (Comparative Example 4).Further, in Comparative Example 4, after the epithelial cells andmesenchymal cells were gently mixed at the ratio of 1:1, one cellaggregate used for generation of a reconstituted tooth germ was preparedin the same way as in Examples 1 to 3.

(3) Histological Analysis

In the case of subrenal capsule transplantation, a reconstituted toothgerm along with surrounding kidney tissue was excised on the 7^(th) dayor 14^(th) day after the transplantation, decalicified with 4.5% EDTA(pH 7.2) for 24 hours after being fixed with a 4%paraformaldehyde-phosphate buffer solution for 6 hours, and embedded inparaffin by a conventional method to produce a 10 μm section of areconstituted tooth germ. For histological analysis, hematoxylin-eosinstaining was performed according to a conventional method.

When a tooth germ derived from a GFP mouse was used for a reconstitutedtooth germ, the tooth germ was deashed with 4.5% EDTA (pH 7.2) for 24hours after being fixed in a 50% (w/v) sucrose-4%paraformaldehyde-phosphate buffer solution for 18 hours, embedded in anOCT compound (Miles Inc., Naperville, Ill.), and 10 μm sections weremade with Cryostat (Leica, Wetzlar, Germany) to be observed under afluorescence microscope (manufactured by Zeiss).

The results from the culture of the entire tooth germ tissue are shownin FIG. 3 and the results from the culture according to the generationmethod of the present invention are shown in FIGS. 4 to 7.

In Comparative Example 1, the entire excised tooth germ was transplantedbeneath a subrenal capsule. As shown in FIG. 3, since the interactionbetween the mesenchymal cells and epithelial cells constituting thetooth germ is not impaired, enamel derived from the epithelial cells anddentin and dental pulp derived from the mesenchymal cells were formed,and a tooth was formed having a tip and root in addition to enamel anddentin arranged in given positions.

On the other hand, as shown in FIGS. 4 to 6, when a single cell formprepared from a tooth germ was used according to the present invention,in other words, when reconstitution was performed by combining toothgerm mesenchymal cells with tooth germ epithelial tissue (Example 1, seeFIGS. 4 and 5) and by combining tooth germ mesenchymal tissue with toothgerm epithelial cells (Example 2, see FIG. 6), a tooth having a specificcell placement with dentin inside and enamel outside was generated bysubrenal capsule transplantation in an 11- to 14-day period. The toothobtained thereby demonstrated that it is possible to reconstitute thesame kind of tooth as that generated normally by culturing an entiretooth germ (FIG. 3).

Furthermore, as shown in FIG. 4, with the present reconstitution andsubrenal capsule transplantation, outer enamel, ameloblast, ameloblast,dentin, and odontoblast were easily identified on the 11^(th) day afterthe transplantation. The root portion was also the same as that fromnormal generation and alveolar bone was identified in the outercircumference of the root portion. Further, from the time courseobservation, dentin and odontoblast were easily identified on the 3^(rd)day after transplantation, and a tooth-specific structure had started toform in the tissue placement. In addition, on the 7^(th) day,accumulation of dentin, odontoblast, and ameloblast were in evidence,and the tooth generation progressed thereafter (data not shown).

Further, immediately after positioning inside a gel drop, it wasobserved under the microscope that cells constituting a cell aggregatewere singly present, and after a single day of short culture, that thecells were strongly bonded and had changed into a single cohesive tissueas in the case of a normal excised tooth germ. This indicated that shortculture prior to transplantation is effective in the formation of tooth.

Moreover, when mesenchymal cells derived from a GFP mouse were used, thecells were localized in dental pulp cells and odontoblasts derived frommesenchymal cells in the inner side (FIG. 5). On the other hand, whenepithelial cells derived from a GFP mouse were used, the cells werelocalized in ameloblasts in the outer side, and the fluorescent imageswere congruous with those of the cell types used (FIG. 6). Therefore, itwas obvious that the same cell interaction was performed as that innormal generation and that reconstitution of tissue was performedwithout sacrificing the directionality of the cells during thegeneration.

In addition, when organ culture was continued without applying subrenalcapsule transplantation, a reconstituted tooth germ which was timecourse cultured from the beginning of the culture, gradually becamelarger, dentin and odontoblast were easily identified on the 16th dayafter the transplantation, and the formation of a tooth-specificstructure was identified in the tissue placement (data not shown). Thiskind of construction by means of organ culture was identified not onlywhen one or the other of epithelial cells and mesenchymal cells was usedas tissue, but also when both were used.

Moreover, when tooth germ epithelial cells and tooth germ mesenchymalcells were used (Example 3), as shown in FIG. 7, the presence of dentinand enamel was confirmed, as when only one of tooth germ epithelialcells and tooth germ mesenchymal cells was used as tissue. When toothgerm epithelial cells and tooth germ mesenchymal cells were used, it wasobserved that plural teeth having directionality and structure werefrequently generated from a single reconstituted tooth germ, suggestingthe possibility of generating plural teeth by separating a tooth budafter generation. In particular, when tooth germ mesenchymal cells werefirst positioned inside a gel drop and then tooth germ epithelial cellswere positioned so as to press against the tooth germ mesenchymal cells,the specific structure, where enamel and dentin are placed outside andinside, respectively, was more precisely constructed and the tooth shapeformed more easily, and it was shown that this may be advantageous intooth formation (data not shown).

Furthermore, when a tooth germ reconstituted by arranging epithelialcells and mesenchymal cells according to the present invention was organcultured, the formation of plural tooth germs and/or tooth buds wasfrequently observed. This suggests the possibility of generating pluralteeth from a single reconstituted tooth germ by surgically separatingthese plural tooth germs and/or tooth buds.

On the other hand, in the case of Comparative Example 2, when culturewas performed with an epithelial tissue alone or an mesenchymal tissuealone, as shown in FIG. 8, a tooth with the specific structure mentionedabove could not be constructed. Therefore, this suggests that tissuehaving the specific structure is reconstituted by performing cellinteraction by the method of the present invention.

Further, in the case of Comparative Example 3, where a low density cellaggregate was used, as shown in FIG. 9, single cells were alreadydispersed in a collagen gel drop during culture and the tooth specificstructure was not reconstituted even by transplanting the cells beneatha subrenal capsule. This suggests that it is preferable to use cells atas high a density as possible in order to reconstitute a tooth by cellinteraction.

Further, in the case of Comparative Example 4, where a cell aggregatewas formed at a high density by mixing tooth germ epithelial cells andtooth germ mesenchymal cells in advance in the ratio of 1:1 withoutcompartmentalization, as shown in FIG. 10, hard tissue such as enameland dentin were not identified. This suggests that it is important toform a cell aggregate compartmentalizing the mass of tooth germepithelial cells and the mass of tooth germ mesenchymal cells, after therespective masses are prepared separately.

(4) Confirmation of Periodontal Tissue

Next, it was ascertained whether or not a tooth formed according to thepresent method had periodontal tissue. In situ hybridization asdescribed below was used to confirm the presence of periodontal tissuein addition to the above-mentioned observation using HE staining images.

A reconstituted tooth germ transplanted beneath a subrenal capsule wasexcised on the 14^(th) day after transplantation, embedded in paraffinby a conventional method, and cut into 10 μm thickness sections. Theparaffin was removed by soaking the sections in a xylene/ethanoldilution series. The sections were treated with 10 μg/ml of Protease K(Nacalai Tesque, Kyoto, Japan) in PBS (−) for 3 minutes and fixed with a4% paraformaldehyde (Nacalai Tesque) phosphate buffer solution for 15minutes. 0.1% (v/v) Triton X-100 (Sigma) was treated in PBS (−) for 3minutes and washed with PBS (−) for 3 minutes. Then they were treatedwith 0.2N HCl (Wako) for 10 minutes and washed with PBS (−) and DEPC(diethyl pyrocarbonate) water respectively for 5 minutes each. Aftertreatment with 1.5% (v/v) triethanol amine (Nacalai Tesque), 0.33N HCl(Wako), and 0.25% (v/v) acetic anhydride (Nacalai Tesque) in DEPC waterfor 10 minutes, the sections were washed twice with 2×SSC for 10minutes. Periostin (Genbank accession No. NM#015784) probe was used byDIG-labeling cDNA section obtained with PCR using sense primer (−7;ggctgaagatggttcctctc, SEQ NO: 1) and antisense primer (573;gtacattgaaggaataacca, SEQ NO: 2). In situ hybridization was performedaccording to a conventional method, colorization was performed with ananti-DIG-Ap Fab fragment (Roche) and an NBT/BCIP Stock Sollution(Roche), and analysis was performed with an Axio Imager A. 1 (Zeiss) andAxioCam Mrc5 (Zeiss).

When the periodontal tissue in the above-mentioned Examples was examinedin detail for the presence or absence of periodontal tissue, alveolarbone similar to that in the normal tooth germ transplantation ofComparative Example 1 (see FIG. 3) was formed around the tooth on the14^(th) day after the transplantation in each of Examples 1 to 3, asshown in FIGS. 4 to 7.

Furthermore, as shown in FIG. 11, despite the combination of singlecells and tissue, alveolar bone and periodontal membrane, which weresimilar to those in the normal tooth germ transplantation of ComparativeExample 1, were formed around the obtained tooth in each of Examples 1to 3. Further, when the tooth of Example 2 was observed, as shown inFIG. 12, expression of periostin mRNA, which is a periodontalmembrane-specific gene, was identified in a region where the formationof periodontal membrane was identified by HE staining (the same wasidentified in Examples 1 and 3).

This indicates that a tooth germ prepared as in Examples 1 to 3 can formperiodontal tissues such as alveolar bone and periodontal membrane.

Examples 4 and 5 Comparative Example 5

After completing the positioning process as described above, organculture commonly used in culture processes was performed continuouslyfor 14 days to analyze tooth generation. A combination of epithelialtissue and mesenchymal cells derived from a tooth germ was used inExample 4, and a combination of epithelial cells and mesenchymal cellsderived from a tooth germ was used in Example 5. In addition, organculture using a normal tooth germ was applied in Comparative Example 5.The results are shown in FIG. 13.

As shown in FIG. 13, in both of Examples 4 and 5, the size of the toothgerm increased as the culture period was lengthened and generation of atooth having a specific structure approximately the same as thatobtained by performing subrenal capsule transplantation was observed.

Moreover, in both of Examples 4 and 5, the teeth obtained from organculture formed a set made up of plural teeth (for example, six teethwhen mesenchymal cells and epithelial cells were used; FIG. 13, lowestrow).

Examples 6 and 7 Comparative Example 6

As shown in Example 5, a tooth obtained from a reconstituted tooth germwas reinduced to form plural teeth despite the fact that the mesenchymalcells and the epithelial cells were prepared from a single reconstitutedtooth germ. Analysis was performed to see whether each tooth reinducedsimultaneously by the reconstituted tooth germ can grow into a singletooth.

(1) Analysis of Separation of Plural Tooth Germs Generated fromReconstituted Tooth Germs and Tooth Generation Potential

1) Individual Separation and Organ Culture of Tooth Germs GeneratedPlurally

A reconstituted tooth germ obtained in a similar way to in Example 3 wasorgan cultured for 2 to 5 days, and plural tooth germs were generatedfrom a single reconstituted tooth germ. Then, on the 2^(nd) day to5^(th) day of the organ culture, single tooth germs were surgicallyseparated from the reconstituted tooth germ that had generated pluraltooth germs using an injection needle and tweezers under astereomicroscope.

Gel drops were prepared by dropping 30 μL of Cellmatrix type I-A (Nittagelatin, Osaka, Japan) onto a silicon grease coated Petri dish in thesame way as in Example 1. Each of the above-mentioned individuallyseparated tooth germs was placed inside a gel drop, and set still in aCO₂ incubator for 10 minutes to solidify the Cellmatrix type I-A (NittaGelatin). Each of the individually separated tooth germs, along with thesurrounding gel, which was a support carrier, was transferred onto themembrane of a cell culture insert in a culture vessel, in which the cellculture insert (PET membrane of pore size of 0.4 micron; BD, FranklinLakes, N.J.) was set so as to contact with DMEM (Sigma) supplemented by10% FCS (JRH), and organ cultured for 18 to 24 hours.

2) Histological Analysis

After culture, each of the individually separated tooth germs along withthe surrounding gel was transplanted beneath a subrenal capsule of an 8week-old C57BL/6 mouse and the individually separated tooth germs wereexcised on the 14^(th) day after the transplantation along with thesurrounding kidney tissue. After the tissue was fixed with a 4%paraformaldehyde-phosphate buffer solution for 6 hours, the tissue wasembedded in paraffin by a conventional method to make a 10 μm section.For histological analysis, hematoxylin-eosin staining was performedaccording to a conventional method.

3) Results

The results of the histological analysis of the separated tooth germs,which were transplanted beneath a subrenal capsule to be generated for14 days, are shown in FIG. 14. As shown in FIG. 14, each of thetransplanted separated tooth germs was developed into a single toothcharacterized by enamel, dentin, dental pulp, a crown and a root.Further, in the obtained teeth, the presence of enamel and dentin in thecrown portion (see “a” on the middle and lower rows of FIG. 14) and anopening of the root in the root portion (see “b” on the mid row of FIG.14) were observed.

These observations indicate that: ameloblast and odontoblast are presentin the crown portion as in a normally generated tooth; the tooth has thesame configuration as that of a normally generated tooth; and each toothgenerated simultaneously as one of a set of teeth is the same as anormally generated tooth in terms of cell placement and directionality.

(2) Tooth Generation by Transplantation of a Reconstituted Tooth Germinto an Oral Cavity

1) Generation of Individually Separated Tooth Germs and IndividuallySeparated Teeth

Individually separated tooth germs were prepared from a reconstitutedtooth germ, from which plural tooth germs were generated, on the 2^(nd)day to 5^(th) day of organ culture as described above. Further, pluralteeth generated from the reconstituted tooth germ, which wastransplanted beneath a subrenal capsule and excised after 14 days oftransplantation, were surgically separated individually using aninjection needle and tweezers under a stereomicroscope.

In the case of an individually separated tooth germ, a gel drop wasprepared by dropping 30 μL of Cellmatrix type I-A (Nitta gelatin, Osaka,Japan), which was prepared at a concentration of 2.4 mg/ml in theabove-mentioned culture solution, on to a silicon grease coated Petridish as described above. The above-mentioned individually separatedtooth germ was placed inside this gel drop, set still in a CO₂ incubatorfor 10 minutes to solidify the Cellmatrix type I-A (Nitta Gelatin).Next, the individually separated tooth germ along with the surroundinggel as a support carrier, was transferred onto the membrane of a cellculture insert in a culture vessel, in which the cell culture insert(PET membrane of pore size of 0.4 micron; BD, Franklin Lakes, N.J.) wasset so as to be in contact with DMEM (Sigma) supplemented by 10% FCS(JRH), and organ cultured for 18 to 24 hours.

After the culture, the surrounding gel was surgically removed with aninjection needle and tweezers, and the individually separated tooth germwas transplanted into a mandibular incisor extraction hole of an 8week-old C57BL/6 mouse; and this was designated as Example 6. Further, atooth individually separated from the reconstituted tooth germ which hadbeen transplanted beneath a subrenal capsule was transplanted to amandibular incisor extraction hole of an 8 week-old C57BL/6 mousewithout being embedded in a gel after separation; and this wasdesignated as Example 7.

2) Methods of Tooth Extraction of an Incisor and Transplantation into anOral Cavity

3 days before transplantation into an oral cavity, an 8 week-old C57BL/6mouse anesthetized with inhaled diethyl ether was injectedintraperitoneally with a physiological salt solution containing 5 mg/mlpentobarbital sodium at a ratio of 200 μl to every 20 g of body weight.A mandible near the eruption site of a mandibular incisor of the mouse,whose sense of pain had been numbed, was exfoliated with a scalpel and atip of the incisor embedded in the jaw bone was exposed. An incisor wasextracted from the mandible using tweezers, blood was wiped off withabsorbent cotton, and bleeding was arrested. For the sake of foodingestion, only the mandibular incisor on one side was extracted andcomminuted feed for breeding was given every day.

An 8 week-old C57BL/6 mouse, whose tooth had been extracted by theabove-mentioned method, was anesthetized with inhaled diethyl ether anda physiological salt solution containing 5 mg/ml pentobarbital sodiumwas injected intraperitoneally at a ratio of 200 μl to every 20 g ofbody weight. The mouse, whose sense of pain was numbed, was fixed on adissecting table such that the side of the jaw, from which the tooth wasextracted, would face up, and the mandible was exposed by cutting theskin and muscle layer from the side of the head in the area of the rootportion of the hole left by the extracted tooth. A hole with a diameterof 1 mm in the case of an individually separated tooth germ and a holewith a diameter of 2 mm in the case of an individually separated toothwas made with a scalpel on the mandible covering the area of the rootportion of the hole left by the extracted tooth, then the individuallyseparated tooth germ or the individually separated tooth wastransplanted into the area of the root portion of the hole left by theextracted tooth through the hole made with the scalpel. The orientationof the individually separated teeth germ or individually separated toothto be transplanted was aligned with that of a normally generated toothand also with the directionality of enamel and periodontal membrane seenin the mandibular incisors of adult mise. The cut muscle layer and skinwere stitched up by a conventional method. The 8 week-old C57BL/6 mousethat had received the oral cavity transplantation was fed withcomminuted feed for breeding every day.

In addition, Comparative Example 6 was designated as an example in whichtransplantation was not performed on a mouse was not transplanted.

3) Histological Analysis

The mandibles to which an individually separated tooth germ andindividually separated tooth had been transplanted were excised on the14^(th) day after the oral cavity transplantation. The bone was deashedwith 22.5% formic acid for 72 hours after being fixed with a 4%paraformaldehyde-phosphate buffer solution for 16 hours; then embeddedin paraffin by a conventional method to make 10 μm sections. 50 ml ofdeashing solution was used for every two mandibles and the whole amountwas replaced at the 48^(th) hour of deashing. For the histologicalanalysis, hematoxylin-eosin staining was performed according to aconventional method.

When a tooth germ derived from a C57BL/6 TgN (act-EGFP) OsbC14-Y01-FM131mouse was used for an individually separated tooth germ and individuallyseparated tooth, it was deashed with 22.5% formic acid for 72 hoursafter being fixed with a 4% paraformaldehyde-phosphate buffer solutionfor 16 hours, embedded in an OCT compound (Miles Inc., Naperville, Ill.)according to a conventional method, and 10 μm sections were made withCryostat (Leica, Wetzlar, Germany) to be observed under a fluorescencemicroscope (Zeiss).

4) Results

Histological pictures on the 14^(th) day after the tooth extraction of amouse in Comparative Example 6, in which transplantation was notperformed on the mouse after the extraction of the incisor, are shown inFIG. 15, and histological pictures of an individually separated toothgerm (Example 6) and an individually separated tooth (Example 7) on the14^(th) day after transplantation into the hole left by an extractedincisor are shown in FIGS. 16 and 17, respectively.

As shown in FIG. 15, in the non-transplanted mouse of ComparativeExample 6, only infiltrated cells and generated bone were identified anda tooth having hard tissue was not identified in a place correspondingto the transplantation site at the hole left by the extracted incisor.

On the other hand, as shown in FIG. 16, at the aforementioned site wherean individually separated tooth germ was transplanted in Example 6, atooth having enamel outside and dentin inside was generated. Thegenerated tooth had a tooth tip and root, the directionality of a tooth,and the same structure as that of a normally generated tooth.

Moreover, in the mouse of Example 7, in which a tooth separated aftergeneration due to subrenal capsule transplantation was transplanted intoa hole left by an extracted tooth, as shown in FIG. 17, a tooth havingenamel outside and dentin inside was generated at the aforementionedsite. The generated tooth had a tooth tip and root, blood vessels insidedental pulp as well as periodontal membrane and alveolar bone around thetooth, and the same structure as that of a normally generated tooth.

Example 8 and Comparative Example 7

(1) Reconstitution of Hair Follicle

In order to demonstrate that the technology developed in the presentinvention is useful in formation of other organs as well as contributingto generation of a tooth germ, reconstitution of a hair follicle wasperformed. Mice were used as models for this experiment.

1) Method of Separating Cells

A hair follicle tissue of a maxillary whisker was excised from an embryoof fetal age of 14.5 days of a C57BL/6N mouse (purchased from CLEAJapan, Inc.) or a C57BL/6-TgN (act-EGFP) OsbC14-Y01-FM131 (RIKENBioresource Center) which is a Green Fluorescence Protein (EGFP)transgenic mouse, under a microscope by a conventional method. The hairfollicle tissue of the maxillary whisker was washed with a phosphatebuffer solution (PBS (−)) containing neither Ca²⁺ nor Mg²⁺, treated withan enzyme solution in which Dispase II (Roche, Mannheim, Germany) at afinal concentration of 1.2 U/ml had been added to the PBS (−), at roomtemperature for 60 minutes, and then washed three times with DMEM(Sigma, St. Louis, Mo.) to which 10% of FCS (JRH Biosciences, Lenexa,Kans.) had been added. Furthermore, DNase I solution (Takara, Shiga,Japan) was added to make a final concentration of 70 U/ml to dispersethe hair follicle tissue, and the hair follicle epithelial tissue andthe hair follicle mesenchymal tissue were surgically separated, using a25 G injection needle (Terumo, Tokyo, Japan).

For hair follicle epithelial cells, the hair follicle epithelial tissueobtained above was washed three times with PBS (−), and treated twicewith an enzyme solution in which Collagenase I (Worthington, Lakewood,N.J.) at a final concentration of 100 U/ml was dissolved in the PBS (−),at 37° C. for 20 minutes. Cells precipitated and retrieved bycentrifugation were further treated with 0.25% Trypsin (Sigma)-PBS (−)at 37° C. for 5 minutes. After washing the cells three times with DMEMsupplemented by 10% FCS, DNase I solution at a final concentration of 70U/ml was added to the cells, and single hair follicle epithelial cellswere obtained by pipetting.

On the other hand, for hair follicle mesenchymal cells, the hairfollicle mesenchymal tissue was washed three times with PBS (−) andtreated with PBS (−) containing 0.25% Trypsin (Sigma) and 50 U/ml ofCollagenase I (Worthington). 70 U/ml of DNase I (Takara) was added andsingle hair follicle mesenchymal cells were obtained by pipetting.

2) Method of Generating Reconstituted Hair Follicle

Next, cells used in generation of a reconstituted hair follicle wereprepared in the same way as in Example 1, except that the hair follicleepithelial cells and hair follicle mesenchymal cells prepared above wereused; 0.2 to 0.3 μL of each of the cells were applyed to a collagen geldrop to prepare respective cell aggregates; and a reconstituted hairfollicle was generated by positioning both cell aggregates in closecontact with each other.

3) Subrenal Capsule Transplantation

For the reconstituted hair follicle generated in a gel, as in Example 1,the cell aggregates, together with the surrounding gel as a supportcarrier, were transferred onto a membrane of a cell culture insert in aculture vessel and organ cultured for 18 to 48 hours. After the organculture, these were transplanted beneath a subrenal capsule of an 8week-old C57BL/6 mouse to promote ectopic hair growth, and the hairgrowth was analyzed.

On the other hand, in Comparative Example 7, a single cell aggregate wasprepared by mixing two types of cells ex vivo in the same way as inComparative Example 4 except that hair follicle epithelial cells andhair follicle mesenchymal cells were used, and this was transplantedbeneath a subrenal capsule as in Example 8.

4) Histological Analysis

In the case of subrenal capsule transplantation, a reconstituted hairfollicle was excised along with the surrounding kidney tissue on the14^(th) day after the transplantation. In organ culture, the cellaggregate was retrieved on the 14^(th) day of the culture. Then, thetissue or cell aggregate was fixed with a 4% paraformaldehyde phosphatebuffer solution for 6 hours, and embedded in paraffin by a conventionalmethod to make 10 μm sections. For histological analysis,hematoxylin-eosin staining was performed according to a conventionalmethod.

5) Results

The results of the subrenal capsule transplantation of a hair folliclein the same type of mouse according to Example 8 are shown in FIG. 18.As shown in FIG. 18, a follicle, inner root sheath, and outer rootsheath derived from epithelial cells and hair papilla cells derived frommesenchymal cells were identified on a longitudinal section of a hairfollicle (section A) since the cell interaction between the epithelialcells and mesenchymal cells which constitute the initial hair folliclewas not impaired when the reconstituted hair follicle was transplanted.Furthermore, in section A, although hair dissolved at the time of tissuestaining, hair which was not completely dissolved was identified. On thecross section (section B), cell placement of an internal root sheath andexternal root sheath was identified such that epithelial cells mightenclose pores. Since hair was dissolved at the time of tissue staining,the residue of hair dissolution was identified.

Moreover, as shown in FIG. 19, on an explant which was excised on the14^(th) day after the subrenal capsule transplantation of areconstituted hair follicle, a hair grown from a hair follicle wasidentified.

On the other hand, in the case of Comparative Example 7, whereepithelial cells and mesenchymal cells were mixed beforehand andreconstituted in a support carrier, a hair follicle tissue was notidentified, as shown in FIG. 20.

Therefore, according to Example 8, hair could be generated from hairfollicle tissue similarly to the cases in which a tooth was generated byusing a tooth germ in Examples 1 to 7.

Thus, according to the present invention, it is shown that celldifferentiation can be effectively induced and tissue havingtissue-specific cell placement and directionality can be generated, bypreparing epithelial tissue/cells and mesenchymal tissue/cellsseparately so that the interaction between the epithelial cells and themesenchymal cells may effectively be performed and by compartmentalizingthe tissue/cells and culturing them in contact with each other at highdensity, and not only for teeth and hair.

Therefore, according to the present invention, it is possible toartificially produce tissue constructed by cell interaction, becausetissue can be reconstructed from various single cells without impairingcell interaction.

EXPLANATION OF LETTERS AND NUMERALS

-   10 gel pack (support carrier)-   12 cell aggregate (a first cell mass)-   14 cell aggregate (a second cell mass)-   16 pipette tip

1. A method of producing a tooth, comprising: positioning a first cellmass substantially containing only either one of mesenchymal cells orepithelial cells, wherein at least one of the mesenchymal cells or theepithelial cells is derived from a tooth germ, and a second cell masssubstantially containing only the other one of the mesenchymal cells orthe epithelial cells, inside a support carrier, such that the first andsecond cell masses are in a state of close contact with each otherinside the support carrier without mixing between the cell masses,wherein the mesenchymal cells or the epithelial cells are in closecontact with each other in the corresponding cell mass; positioning thefirst cell mass inside the support carrier such that the second cellmass makes a depression in the first cell mass such that the first cellmass substantially envelops the second cell mass when the second cellmass is inserted into the support carrier; and culturing the first andthe second cell masses inside the support carrier until the tooth isformed, while maintaining the state of close contact between the cellmasses.
 2. The method of producing a tooth according to claim 1, whereinboth the mesenchymal cells and the epithelial cells are derived from atooth germ.
 3. The method of producing a tooth according to claim 1,further comprising a first preparation process of preparing the firstcell mass and a second preparation process of preparing the second cellmass prior to positioning the cell masses in contact with each other. 4.The method of producing a tooth according to claim 1, comprisingconducting growth inside the support carrier in the presence of othercells of animals.
 5. The method of producing a tooth according to claim1, wherein each of the first cell mass and the second cell mass is acell mass of single cells.
 6. A method of producing a tooth withperiodontal tissue comprising: positioning a first cell masssubstantially containing only either one of mesenchymal cells orepithelial cells, wherein at least one of the mesenchymal cells or theepithelial cells is derived from a tooth germ, and a second cell masssubstantially containing only the other one of the mesenchymal cells orthe epithelial cells, inside a support carrier, such that the first andthe second cell masses are in a state of close contact with each otherinside the support carrier without mixing between the cell masses,wherein the mesenchymal cells or the epithelial cells are in closecontact with each other in the corresponding cell mass; positioning thefirst cell mass inside the support carrier such that the second cellmass makes a depression in the first cell mass such that the first cellmass substantially envelops the second cell mass when the second cellmass is inserted into the support carrier; culturing the first and thesecond cell masses inside the support carrier until the tooth is formed,while maintaining the state of close contact between the cell masses;and continuing the culturing until the periodontal tissue is formed. 7.A method of producing periodontal tissue, comprising: positioning afirst cell mass substantially containing only either one of mesenchymalcells or epithelial cells, wherein at least one of the mesenchymal cellsor the epithelial cells is derived from a tooth germ, and a second cellmass substantially containing only the other one of the mesenchymalcells or the epithelial cells, inside a support carrier, such that thefirst and second cell masses are in a state of close contact with eachother inside the support carrier without mixing between the cell masses,wherein the mesenchymal cells or the epithelial cells are in closecontact with each other in the corresponding cell mass; positioning thefirst cell mass inside the support carrier such that the second cellmass makes a depression in the first cell mass such that the first cellmass substantially envelops the second cell mass when the second cellmass is inserted into the support carrier; culturing the first and thesecond cell masses inside the support carrier until a tooth andperiodontal tissue contiguous to the tooth are obtained, whilemaintaining the state of close contact between the cell masses; andisolating the periodontal tissue obtained by the culturing.
 8. A set ofteeth, obtained by positioning a first cell mass substantiallycontaining only either one of mesenchymal cells or epithelial cells,wherein at least one of the mesenchymal cells or the epithelial cells isderived from a tooth germ, and a second cell mass substantiallycontaining only the other one of the mesenchymal cells or the epithelialcells, inside a support carrier, such that the first and the second cellmasses are in a state of close contact with each other inside thesupport carrier without mixing between the cell masses, wherein themesenchymal cells or the epithelial cells are in close contact with eachother in the corresponding cell mass; positioning the first cell massinside the support carrier such that the second cell mass makes adepression in the first cell mass such that the first cell masssubstantially envelops the second cell mass when the second cell mass isinserted into the support carrier; and culturing the first and thesecond cell masses inside the support carrier until the set of teeth isformed, while maintaining the state of close contact between the cellmasses.
 9. The set of teeth according to claim 8, wherein both themesenchymal cells and the epithelial cells are derived from a toothgerm.
 10. A method of producing a tissue that is constructed by aninteraction between mesenchymal cells and epithelial cells, the methodcomprising: positioning a first cell mass substantially containing onlyeither one of mesenchymal cells or epithelial cells and a second cellmass substantially containing only the other one of the mesenchymalcells or the epithelial cells, inside a support carrier, such that thefirst and the second cell masses are in a state of close contact witheach other inside the support carrier without mixing between the cellmasses, wherein the mesenchymal cells or the epithelial cells are inclose contact with each other in the corresponding cell mass;positioning the first cell mass inside the support carrier such that thesecond cell mass makes a depression in the first cell mass such that thefirst cell mass substantially envelops the second cell mass when thesecond cell mass is inserted into the support carrier; and culturing thefirst and the second cell masses inside the support carrier until thetissue is formed, while maintaining the state of close contact betweenthe cell masses.
 11. The method of producing a tissue according to claim10, further comprising a first preparation process of preparing thefirst cell mass and a second preparation process of preparing the secondcell mass prior to positioning the cell masses in contact with eachother.
 12. The method of producing a tissue according to claim 10,wherein at least one of the mesenchymal cells and the epithelial cellsis derived from a targeted tissue.
 13. The method of producing a tissueaccording to claim 12, wherein the targeted tissue is selected from thegroup consisting of a tooth, a hair, a kidney, a lung and a liver.
 14. Amethod of treatment for tooth loss or damage, comprising: transplantingat least one tooth selected from (i) a tooth obtained by the method ofproducing a tooth according to claim 1, and (ii) a tooth withperiodontal tissue produced at the site of tooth loss or damage by themethod of: (a) positioning a first cell mass substantially containingonly either one of mesenchymal cells or epithelial cells, wherein atleast one of the mesenchymal cells or the epithelial cells is derivedfrom a tooth germ, and a second cell mass substantially containing onlythe other one of the mesenchymal cells or the epithelial cells, inside asupport carrier and in contact with each other; and (b) culturing thefirst and the second cell masses inside the support carrier until atooth and periodontal tissue contiguous to the tooth are obtained.
 15. Amethod of treatment for tooth loss or damage, comprising: transplantingat least one tooth selected from (i) a tooth obtained by the method ofproducing a tooth according to claim 2, and (ii) a tooth withperiodontal tissue produced at the site of tooth loss or damage by themethod of: (a) positioning a first cell mass substantially containingonly either one of mesenchymal cells or epithelial cells, wherein atleast one of the mesenchymal cells or the epithelial cells is derivedfrom a tooth germ, and a second cell mass substantially containing onlythe other one of the mesenchymal cells or the epithelial cells, inside asupport carrier and in contact with each other; and (b) culturing thefirst and the second cell masses inside the support carrier until atooth and periodontal tissue contiguous to the tooth are obtained.
 16. Amethod of treatment for tooth loss or damage, comprising: transplantingat least one tooth selected from (i) a tooth obtained by the method ofproducing a tooth according to claim 3, and (ii) a tooth withperiodontal tissue produced at the site of tooth loss or damage by themethod of: (a) positioning a first cell mass substantially containingonly either one of mesenchymal cells or epithelial cells, wherein atleast one of the mesenchymal cells or the epithelial cells is derivedfrom a tooth germ, and a second cell mass substantially containing onlythe other one of the mesenchymal cells or the epithelial cells, inside asupport carrier and in contact with each other; and (b) culturing thefirst and the second cell masses inside the support carrier until atooth and periodontal tissue contiguous to the tooth are obtained.
 17. Amethod of treatment for tooth loss or damage, comprising: transplantingat least one tooth selected from (i) a tooth obtained by the method ofproducing a tooth according to claim 4, and (ii) a tooth withperiodontal tissue produced at the site of tooth loss or damage by themethod of: (a) positioning a first cell mass substantially containingonly either one of mesenchymal cells or epithelial cells, wherein atleast one of the mesenchymal cells or the epithelial cells is derivedfrom a tooth germ, and a second cell mass substantially containing onlythe other one of the mesenchymal cells or the epithelial cells, inside asupport carrier and in contact with each other; and (b) culturing thefirst and the second cell masses inside the support carrier until atooth and periodontal tissue contiguous to the tooth are obtained.
 18. Amethod of treatment for tooth loss or damage, comprising: transplantingat least one tooth selected from (i) a tooth obtained by the method ofproducing a tooth according to claim 5, and (ii) a tooth withperiodontal tissue produced at the site of tooth loss or damage by themethod of: (a) positioning a first cell mass substantially containingonly either one of mesenchymal cells or epithelial cells, wherein atleast one of the mesenchymal cells or the epithelial cells is derivedfrom a tooth germ, and a second cell mass substantially containing onlythe other one of the mesenchymal cells or the epithelial cells, inside asupport carrier and in contact with each other; and (b) culturing thefirst and the second cell masses inside the support carrier until atooth and periodontal tissue contiguous to the tooth are obtained.
 19. Amethod of treatment for tooth loss or damage, comprising: transplantingat least one tooth selected from (i) a tooth obtained by the method ofproducing a tooth according to claim 6, and (ii) a tooth withperiodontal tissue produced at the site of tooth loss or damage by themethod of: (a) positioning a first cell mass substantially containingonly either one of mesenchymal cells or epithelial cells, wherein atleast one of the mesenchymal cells or the epithelial cells is derivedfrom a tooth germ, and a second cell mass substantially containing onlythe other one of the mesenchymal cells or the epithelial cells, inside asupport carrier and in contact with each other; and (b) culturing thefirst and the second cell masses inside the support carrier until atooth and periodontal tissue contiguous to the tooth are obtained. 20.The method of producing a tooth with periodontal tissue according toclaim 6, wherein both the mesenchymal cells and the epithelial cells arederived from a tooth germ.
 21. The method of producing a tooth withperiodontal tissue according to claim 6, further comprising a firstpreparation process of preparing the first cell mass and a secondpreparation process of preparing the second cell mass prior topositioning the cell masses in contact with each other.
 22. The methodof producing a tooth with periodontal tissue according to claim 6,comprising conducting growth inside the support carrier in the presenceof other cells of animals.
 23. The method of producing a tooth withperiodontal tissue according to claim 6, wherein each of the first cellmass and the second cell mass is a cell mass of single cells.